Evaluation of Nested broad-range PCR for Pathogen Detection in Negative Blood Cultures

Abstract

Background: Bloodstream infections are a major cause of morbidity and mortality among all age groups. In the laboratory, it is routinely diagnosed by performing a blood culture which is considered an imperfect gold standard. The diagnostic limitations and uncertainties of blood cultures are related to low sensitivity, particularly in antibiotic-exposed cases, and extended time for pathogen detection that increases turn-around-time of the results. An alternative and recently introduced nucleic acid amplification method for 16S rDNA, a conserved DNA sequence common to all bacteria, has gained significant. It has high sensitivity and an ability to detect organisms that are non-cultivable or nonviable owing to prior antibiotic treatment and where the organism fails to grow on a culture medium. Objectives: To determine the utility of broad-range PCR in the detection of pathogens from negative blood culture was the aim of our study. Materials and Methods: A total of 50 negative blood cultures after 5 days of incubation on BacT/Alert continuously monitored blood culture system were included in this study. From each blood broth sample, DNA was isolated which was subjected to a broad range 16S rDNA nested PCR. The PCR products obtained were visualized on agarose gel electrophoresis and the results were interpreted. Results: Broad range bacterial 16S rDNA nested PCR detected the desired amplicon in 4/50 (8%) of the blood culture-negative samples. Conclusion: The broad range bacterial nested PCR method if used in combination with routine culture techniques in clinical microbiology laboratories will increase the detection of Bloodstream pathogens.